Prion protein (PrP) underlies a spectrum of diseases with no established treatment and devastating human and economic consequences. Several unique features separate prion diseases from other neurodegenerative maladies. (i) There is an infectious isoform of the prion protein, PrPSc, that propagates its abnormal conformation in an autocatalytic manner using the normal isoform, PrPC, as a substrate. (ii) The process of propagation requires the amino acid sequences of PrPC and PrPSc to be identical (or highly homologous). (iii) Within the same primary structure, PrP is capable of adopting conformationally distinct states when it is converted into pathological isoforms, which are known as "strains" of PrPSc. These features indicate that prion diseases have a unique molecular mechanism, distinct from other disorders related to protein aggregation. Our general strategy is to concentrate on these features of PrPSc propagation as a key for understanding the mechanism of prion disease. The hypothesis to be tested is whether these distinguishing features are inherent properties of the prion protein. In particular, the PI will examine the extent to which the process of aggregation of non-glycosylated recombinant PrP mimics basic hallmarks of prion diseases outside of the cellular environment. Using state-of-the-art biochemical and biophysical methods, three aspects of the self-propagating process will be investigated: (1) the kinetic pathway of self-propagating aggregation, with special emphasis on the early events and the rate-limiting step of conversion; (2) the biophysical nature of the autocatalytic mechanism of aggregation; (3) the conformational diversity of self-propagating aggregates and fidelity of propagation. In the proposed work, an array of biophysical tools will be used: proteinase K digestion combined with mass spectrometry, immunoconformational assay, analytical size-exclusion chromatography with unique ultra sensitive detection, spectroscopic techniques, dynamic light scattering, and electron microscopy. The proposed research work will shed light on the mechanism of self-propagation, will reveal novel rules of protein folding that govern specific aggregations, and define the features that make PrP unique among proteins. Such knowledge should lay the foundation for novel molecular and pharmacological approaches for treating prion diseases.